Structure-Informed Design of an Ultra Bright RNA-activated Fluorophore

Fluorogenic RNAs such as the Mango aptamers are uniquely powerful tools for imaging RNA. A central challenge has been to develop brighter, more specific, and higher affinity aptamer-ligand systems for cellular imaging. Here, we report an ultra-bright fluorophore for the Mango II system discovered using a structure-informed, fragment-based small molecule microarray approach. The new dye, Structure informed, Array-enabled LigAnD 1 (SALAD1) exhibits 3.5-fold brighter fluorescence than TO1-Biotin and subnanomolar aptamer affinity. Improved performance comes solely from alteration of dye-RNA interactions, without alteration of the chromophore itself. Multiple high-resolution structures reveal a unique and specific binding mode for the new dye resulting from improved pocket occupancy, a more defined binding pose, and a novel bonding interaction with potassium. The dye notably improves in-cell confocal RNA imaging. This work provides both introduces a new RNA-activated fluorophore and also a powerful demonstration of how to leverage fragment-based ligand discovery against RNA targets.


INTRODUCTION
The development of RNA aptamers that uoresce when bound to small molecule dyes has shown great potential in the eld of RNA imaging. 1,2 inding between dyes and RNA aptamers signi cantly enhances uorescence yielding excellent signal to noise ratios.Many different uorogenic RNA aptamers have now been developed, including the malachite green aptamer, Spinach, Broccoli, Mango, Corn, and Pepper, thio avin T-based systems, 3 PNA-based probes, 4 and related variants. 5,6,7 Mre recently, uorogenic DNA aptamers have been reported. 8In general, these aptamers bind to a variety of small molecule dyes, some inspired by the green uorescent protein chromophore, to induce greatly enhanced uorescence of the dye. 9Aptamers have been developed that uoresce across a wide range of emission wavelengths ranging from 500 nm to 660 nm. 6Fluorogenic aptamers have been used in diverse cellular imaging studies, including multi-color RNA imaging and single-molecule spectroscopy. 10,11,12,13,14 In ddition to their use as imaging agents, uorogenic aptamers have been leveraged to create sensors. 15,16 he fact that these aptamers are genetically encodable, coupled with exibility in choicd of excitation-emission wavelengths makes them powerful tools for diverse applications.
Among the above-mentioned RNA aptamers, Mango is a well characterized system that binds tightly to thiazole orange (TO) and related dyes. 17To date, four generations of Mango aptamers have been developed, all exhibiting low nanomolar a nities to TO derivatives including TO1-Biotin and TO3-biotin. 18In-depth structural studies including X-ray crystallography have revealed that the aptamers fold into a complex G-quadruplex-containing structure and accomplish turn-on uorescence by constraining the TO uorophore into a planar conformation. 17,19,20,21 Th Mango II aptamer contains a well-de ned but plastic pocket capable of binding TO derivatives in multiple orientations.To date, most work on the Mango system has focused on evolution of improved aptamers, 22,23 development of uorogenic transcripts for live cell or single molecule imaging, 24 or rational modi cation of the TO uorophore to alter excitation/emission pro les. 25 The discovery of Mango aptamers represents a powerful advance in the development of RNA imaging tools.
The existence of high-quality structural information coupled with uniquely valuable imaging applications makes the Mango aptamers an attractive system for structure-informed ligand design against RNA.Recognition of RNA as an important target for small molecules is increasing. 26,27,28,29 Hoever, strategies to develop potent, selective small molecule ligands for RNA still lag far behind protein targeting strategies.One approach that has gained some attention is fragment-based design.Fragment-based drug design enables the rapid development of tight and speci c small molecule binders for a target from weak but highly speci c low molecular weight ligands. 30,31 n recent years, fragment-based technologies have been broadly applied to RNA, 32 including nuclear magnetic resonance (NMR), 33,34 equilibrium dialysis, 35 selective 2'-hydroxyl acylation analyzed by primer extension and mutational pro ling (SHAPE-MaP), 36 surface plasmon resonance (SPR), 37 mass spectrometry (MS), 38 and DNA encoded library (DEL)-based screening. 39Upon performing a ligandability analysis of the Mango II TO-binding pocket, we reasoned that a fragment-based screening approach could be an appropriate pathway to develop new uorescent RNA ligands with improved properties.
Here, we report the structure-informed discovery of a new generation of TO-derived uorophores for the Mango II RNA aptamer using a fragment-based microarray screening strategy.Biophysical analysis of dye-and fragment-RNA interactions revealed non-competitive binding between one identi ed fragment and TO.By linking the fragment and TO, multiple new dyes were created, with sub-nanomolar binding a nities.Critically, one compound (SALAD1) exhibited 3.5-fold improved brightness relative to TO and TO1-Biotin, both of which are typically used in cellular imaging with Mango aptamers.Structure visualization with four different dye-aptamer complexes revealed a unique binding mode distinct from TO1-Biotin.Photophysical analysis and structure-activity relationship studies revealed a critical combination of functional groups necessary for the observed uorescence enhancement.Finally, the improved uorescence yields notable advantages in high-signal confocal cellular imaging.Taken together, this structure-informed approach reveals how fragmentbased ligand design, targeted against an RNA aptamer, can lead to notable enhancement of uorescence and in-cell imaging by constraining the uorophore in a unique binding pose.

High-throughput screening for Mango II RNA-binding fragments
To develop a novel TO-derived uorophore, we rst analyzed the binding pockets for ligands within the Mango aptamers.The binding modes of TO1-based ligands in all four Mango aptamers were compared qualitatively and using ICM MolSoft Pocket nder software (Figure S1).Analysis of the co-crystal structure of TO1-Mango II complex (PDB: 6C63) revealed that the ligand binding pocket is hydrophobic and within acceptable volume for recognizing small molecule ligands.When bound to the aptamer, TO1-Biotin stacks on the guanine tetrads and interacts with A12 and A17, while the biotin sidechain was solvent exposed and not resolved in the crystal structure.
By docking a structure of TO (which lacks the biotin-bearing sidechain) to the Mango II aptamer, we found that an empty space exists within the pocket.We reasoned that this space could accommodate a separate fragment, potentially linked through the methyl group on TO, that could be leveraged to improve binding (Fig. 1A and S2).
To e ciently identify a TO co-binder, we developed fragment microarrays that could be used to screen tagged Mango aptamers in the presence or absence of the TO ligand.Brie y, a total of 2,214 fragments were curated and purchased from Enamine, all of which contained amine and alcohol groups compatible with array manufacture.Fragments were printed onto isocyanate-modi ed glass slides based on previously reported methods for small molecule microarray (SMM) fabrication. 40In parallel, a screening construct consisting of the Mango II RNA tagged with a poly-A tail was annealed with a Cy5-poly(dT) oligonucleotide.This construct was dissolved in a folding buffer containing140 mM KCl, annealed, and analyzed by circular dichroism (CD) to con rm proper aptamer folding.The screening construct exhibited a maximum at 263 nm and a minimum at 240 nm, consistent with a folded, parallel G4 structure.These features were not observed in LiCl buffer (Figure S3).Once proper folding was con rmed, arrays were incubated with the screening construct, and the uorescence intensity for each spot was quanti ed (using Z-scores, across two replicates).In parallel, a second screen was performed in the presence of a saturating concentration (10 equivalents) of TO.Pearson correlation coe cient (r) values for Mango II and Mango II + TO assays were 0.81 and 0.84, respectively, con rming a reproducible screen (Figure S4A).In contrast, comparing the two different screening results yielded a Pearson correlation of 0.04, indicating that distinct sets of compounds scored as hits in the presence versus absence of TO (Fig. 1B).A total of 30 fragments (F1-F30) were identi ed as hits for Mango II RNA (Figure S4B).Binding of 11 fragments was non-competitive with TO (F1-F11, Figure S5), while the remaining 19 were identi ed as competitive (F12-F30, Figure S6).We hypothesized that some of the non-competitive hits may bind to the available pocket in the Mango aptamer (Fig. 1A).

Characterization of Fragment Binding to Mango II
For the 11 fragments showed non-competitive binding behavior with TO by SMM, we explored whether any fragments impacted ligand uorescence.We performed a uorescence intensity assay by titrating fragments into a solution containing the TO-Mango II complex.
Titrations contained a TO concentration of 500 nM so that binding to the Mango II pocket (K D = 100 nM) was fully saturated.
Fluorescence was measured as a function of fragment concentration.Six non-competitive fragments (F1, F2, F3, F5, F6, and F10) signi cantly enhanced the TO uorescence (ranging from 5-116%), while the other ve showed weak or no effects (Fig. 2A).Several fragments (F1-F3) capable of enhancing uorescence contained similar structural chemotype (Figure S7).In addition, competitive binders, including three representative fragments (F28, 29 and 30) and two G4 stacking ligands (BRACO19 and PhenDC3), were also tested and indeed showed competition in uorescence assay, emphasizing our ability to identify co-binding fragments.Among all the fragments, F2 showed the most promising uorescence enhancement behavior with an EC 50 value of 52 ± 19 µM and 95% improvement in uorescence intensity (Fig. 2A and S7), along with high Z scores (Fig. 2B) and was selected for further study.F2 itself is not uorescent, emphasizing that the fragment enhanced the uorescence of TO itself (Figure S8).Together, these observations both validate the ability of SMMs to identify noncompetitive ligands, and also remarkably revealed that noncompetitive and non-covalently binding ligands can enhance the uorescence of TO.
Characterization of F2 as a co-binder with TO to Mango II RNA Binding of F2 to the TO-Mango II complex was characterized by multiple approaches.Surface plasmon resonance (SPR) was performed using polyA-containing Mango II RNA annealed a with biotinylated poly(dT) oligo, immobilized on a streptavidin surface.Injection of F2 (500 µM) or TO (1 µM) yielded binding signals of 10 ± 1 and 41 ± 2 response units (RU), respectively.Injecting both TO and F2 resulted in an observed binding level of 52 ± 4 RU, roughly equivalent to the sum of the response observed for individual components (Fig. 2C), and thus con rming co-binding.Fitting titrations of F2 showed a K D of 700 ± 260 µM.In a parallel experiment, samples were pre-equilibrated with saturating (500 nM) TO and then titrated with F2.In the presence of TO, the dissociation constant remained similar (K D = 450 ± 120 µM), indicating that TO binding to the RNA does not signi cantly impact the K D of F2. (Figure S9).In parallel, uorescence titrations were used to con rm the observation that F2 binding does not in uence the K D of TO for the aptamer.(Figure S10).
Binding of F2 was also evaluated using water ligand observed gradient spectroscopy (waterLOGSY) NMR.Here, positive phasing of the ligand peaks identi es a binding interaction (Fig. 2D).Under these conditions, F2 only bound to Mango II RNA in the presence of TO.In contrast, omitting other components (TO or RNA) led to the negative-phasing NMR spectrum, indicating no interaction.Together, these results con rm that F2 occupies an RNA binding site distinct from TO.
Linking TO and F2 yields a dye with enhanced uorescence Encouraged by the observation that non-covalently bound F2 enhanced the uorescence of TO, we designed and synthesized new uorescent probes by linking TO with F2 and related compounds.We developed a four-step route to synthesize a new dye, consisting of F2 linked to TO through an amide linker adjacent to the benzothiazole ring of TO (Fig. 3A, Supplementary Methods).The new ligand, Structure-informed, Array-enabled LigAnD, was named SALAD1.In addition, we synthesized three additional analogs lacking functional groups on the fragment benzyl ring-one lacking the uorine (SALAD3), one lacking the pyrazole ring (SALAD4), and another lacking both groups (SALAD2) (Fig. 3A, Supplementary Methods).
Relative to TO ( ex = 510 nm, em = 533 nm) and TO1-Biotin ( ex = 510 nm, em = 535 nm), SALAD1 has a similar but slightly redshifted excitation and emission pro le ( ex = 511 nm, em = 540 nm) (Table 1, Fig. 3B).SALAD1 also displayed a slightly larger Stokes shift of 29 nm relative to TO (22 nm) and TO1-Biotin (25 nm).Similar excitation and emission pro les were observed for the other analogs (Figure S11).Relative uorescence intensities of the dyes were compared through a uorescence intensity assay (Fig. 3C).The SALAD1 compound displayed greater than 3.5-fold brighter uorescence than TO and TO1-Biotin at high RNA concentrations.Our other analogs showed lower uorescence intensities than did SALAD1 and TO, emphasizing that all molecular features of F2 are necessary to enhance uorescence.
Further photophysical characterization revealed that SALAD1 displays properties similar to existing TO-based dyes (Table 1).When bound to Mango II, SALAD1 shows a 514-fold uorescence enhancement, compared to 643-and 647-fold turn-on for TO and TO1-Biotin, respectively.The extinction coe cient of SALAD1 (45,422 M − 1 cm − 1 ) is also comparable to the extinction coe cients for TO (53,784 M − 1 cm − 1 ) and TO1-Biotin (77,500 M − 1 cm − 1 ).SALAD3 is the only other analog that displayed similar properties, with a turn-on of 711-fold and an extinction coe cient of 30,235 M − 1 cm − 1 .SALAD2 and SALAD4 showed signi cantly weaker uorescence enhancement values and lower extinction coe cients indicating that the pyrazole ring plays a critical role for these properties.
Apparent K D values were determined for each compound using dose-dependent uorescence intensity assays (Fig. 3C, S12, and Table 1).SALAD1 (K D app = 0.69 ± 0.1 nM) binds 7.5-fold more tightly to Mango II compared to TO (K D app = 5.9 ± 1.4 nM), demonstrating that the new dye had a signi cantly improved binding a nity to the aptamer.The observed binding a nity is comparable to TO1-Biotin (K D app = 0.85 ± 0.2 nM), despite the difference in uorescence intensities.Intriguingly, SALAD2 (K D app = 0.27 ± 0.03 nM), SALAD3 (K D app = 0.29 ± 0.02 nM), and SALAD4 (K D app = 0.21 ± 0.03 nM) all displayed tighter binding a nities to Mango II in uorescence intensity assays, indicating that binding a nity and uorescence intensity are not directly related for this system.
TO is known to bind nonspeci cally to nucleic acid structures, limiting its utility in targeted imaging applications. 41We assessed the selectivity of SALAD1 by measuring uorescence when incubated with representative RNA and DNA structures, including several Gquadruplexes (Table S1).SALAD1 showed changes in uorescence intensity in the presence of all four generations of Mango and uoresced brightest when bound to Mangos II and III (Fig. 3D).In contrast, weaker or no binding was observed to other G4 and with non-G4 nucleic acid structures, indicating selective interactions at relevant concentrations.

X-ray crystal structures reveal unique binding mode of new dyes
We determined co-crystal structures of our new uorophores with the Mango II RNA at 2.85-3.0Å resolution (Table S2, Supplementary Methods).All of the new uorophores bind the aptamer RNA with a 1:1 stoichiometry, and in the same binding pocket as occupied by TO1-Biotin (Figure S13). 19Two of the new ligands (SALAD1 and SALAD3) ll the binding pocket to a larger extent than TO1-Biotin (Fig. 4 and S14).The buried solvent-accessible area for SALAD1 and SALAD3 are 590.4Å 2 and 594.0 Å 2 , respectively, whereas Mango II buried 529.1 ± 5.1 Å 2 (average of two well-resolved crystallographically-independent complexes in the structure ± s.d.)In contrast, SALAD2 and SALAD4 have a less extensive RNA interface than TO1-Biotin (burying 514.6 Å 2 and 514.8 Å 2 , respectively).Regardless of the degree of occupancy of the binding site, the uorophores exhibit multiple binding poses in all of our co-crystal structures.Thus, the new ligands do not completely resolve the binding-site promiscuity originally noted for the Mango II-TO1-Biotin complex (PDB:6C63). 19en TO1-Biotin binds to Mango II, it creates an unoccupied a cavity adjacent to RNA residue A22.This purine nucleotide adopts a similar conformation in the TO1-Biotin complex and in complexes with the new uorophores, except in the SALAD1 complex.In the SALAD1-containing structure, A22 adopts the synglycosidic bond conformation (rather than the anticonformation present in all other Mango II complex structures), and the purine base is displaced to the top of the uorophore (Fig. 4B and S15).Binding of SALAD1 thus results in a substantial rearrangement of the uorophore-binding pocket of the RNA.Further, the carbonyl oxygen of the amide group of SALAD1 is uniquely within coordination distance (3.1 Å) from the K + ion of the adjacent G-quadruplex (at the precision of the current atomic coordinates; Table S2).Altogether, the larger interfacial area, the enhanced interaction with A22 resulting from the RNA conformational change, and the additional metal ion coordination are consistent with the improved properties of SALAD1.
In-cell confocal imaging of Mango II RNA using the improved dye Historically, efforts to improve uorescent aptamers as in-cell imaging tools have focused on altering brightness, photostability, and background signal.Mango II-TO1-Biotin has been used to image RNA localization in cells via single molecule uorescence microscopy, 13 but only limited work has been published using confocal microscopy to image Mango systems, 25,42 likely due to insu cient brightness.
Confocal microscopy enables the capture of high-resolution images of in-focus light, making it a powerful technique for imaging RNA uorogenic aptamers that might otherwise display background uorescence.HEK293T cells were transfected with the previously described 24 mCherry-Mango II x 24 plasmid which allows both protein and RNA expression levels to be monitored in cells in the same imaging experiment.Cells were xed and treated with 2 µM of either SALAD1 or TO1-Biotin and prepared for imaging.In cells with stained with Hoechst dye to image nuclei (Fig. 5A and E), Mango II-containing RNA transcription (Fig. 5B and F) and mCherry expression (Fig. 5C and G) were observed via uorescent imaging.The new SALAD1 dye visually uoresces brighter than TO1-Biotin when bound to Mango II in cells (Figure .5B and F).Additionally, mean uorescence intensity is 3-fold brighter (Figure . 5I).Thus, SALAD1 as a dramatically brighter uorophore, is suitable for confocal imaging and holds notable potential for imaging RNA in whole cells.

CONCLUSIONS
Here, we show that the development of novel RNA-binding ligands with improved properties can be achieved through a fragment-based, structure-informed strategy.The Mango II system is a rare example of a well-characterized RNA-ligand complex and was well-suited to explore our approach.We used an SMM screening platform in concert with a curated library of fragments to discover a fragment that cobinds, non-competitively, with TO to Mango II and increases the uorescence intensity of TO.Covalently linking the fragment to TO resulted in a dye with subnanomolar a nity for the aptamer.Our approach conceptually differs from methods that focus on chemical alteration of the chromophore, and we nd that fragment linking results in unexpected and welcome improvements of diverse uorophore properties, such as high a nity, selectivity, turn-on ratio, and uorescence intensity.
Compared to TO and TO1-Biotin, the dye described here displays much higher uorescence intensity in the presence of Mango II.Remarkably, SALAD1 also uoresces brighter than the other closely related analogs lacking speci c functional groups decorating the benzyl fragment, indicating that both substituents are necessary to achieve high uorescence enhancement.The pyrazole ring in particular seems to play an important role for the turn-on and extinction coe cient values of the synthesized dyes.This nding demonstrates that our screening approach produced a fragment hit with speci c properties that led to the design of the improved dye.
Additionally, while all four SALAD analogs all bound tighter than TO to Mango II, crystal structures revealed that the unique binding pose of SALAD1 and associated conformational effects on the RNA may explain the unique enhanced uorescence properties of the dye.In contrast to the other Mango-dye complex structures, SALAD1 binds to Mango II in a manner that results in a more enclosed binding site through altering the conformation of A22.Not only that, but this binding pose positions the carbonyl oxygen within bonding distance of the stabilizing potassium ion through the center of the guanine tetrad.To the best of our knowledge this is the rst time a small molecule ligand has been observed to position a group within bonding distance of the potassium ion of a G4, a novel bonding interaction that could have broad implications for the design of other G4 ligands.By constraining the uorophore in a unique pose, these new binding features contribute to both the dramatic increase in binding a nity and uorescence intensity of SALAD1, highlighting the utility of our approach for structure-informed ligand design.
The net result of these distinctive features and the speci c molecular interaction of SALAD1 is a new brighter and more practical class of RNA-activated turn-on uorophores.Due to its bright uorescence and low background, the "Mango-SALAD" system is well-suited for confocal imaging and is poised to create opportunities to better study RNA expression and localization.Additionally, this work stands as a powerful demonstration of the potential for structure-informed design and fragment-based discovery to develop novel ligands with improved binding a nity, selectivity, and other properties for RNA targets.Design strategies described herein have implications both for the development of improved imaging probes, including other uorogenic RNA aptamers, as well as for medicinal chemistry efforts to develop biologically active compounds that interact with therapeutically relevant RNAs. Structure

Supplementary Files
This is a list of les associated with this preprint.Click to download. MangoSI06052024MY.docx and fragment-binding to the Mango aptamer.(A) Pocket analysis of Mango II RNA aptamer modeled in presence of TO. (B) Fragment microarray-based screening using Cy5-labeled Mango II RNA (250 nM) with/without competing TO (2.5 µM).Replicate screenings were performed for each sample.Z-score comparison of each fragment as a function of incubation conditions (Mango II vs. Mango II + TO).Fragments bind to the Mango II aptamer in both competitive and non-competitive modes.

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Table 1
Photophysical properties of TO-based uorophores 25Obtained in a previous study25